Journal article
Hydrogen-Exchange Stabilities of RNase T1 and Variants with Buried and Solvent-Exposed Ala → Gly Mutations in the Helix
Biochemistry (Easton), Vol.38(50), pp.16481-16490
12/14/1999
DOI: 10.1021/bi9919450
PMID: 10600109
Abstract
Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala → Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen- exchange stabilities (ΔG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (ΔG(U)), if the effects of D2O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in ΔG(HX) upon Ala → Gly mutations (ΔΔG(HX)) as compared to equilibrium measurements (ΔΔG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833- 2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala → Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.
Details
- Title: Subtitle
- Hydrogen-Exchange Stabilities of RNase T1 and Variants with Buried and Solvent-Exposed Ala → Gly Mutations in the Helix
- Creators
- Beatrice M. P. HUYGHUES-DESPOINTES - Texas A&M UniversityUlrike Langhorst - Vrije Universiteit BrusselJan Steyaert - Vrije Universiteit BrusselC. Nick Pace - Vrije Universiteit BrusselJ. Martin Scholtz - Vrije Universiteit Brussel
- Resource Type
- Journal article
- Publication Details
- Biochemistry (Easton), Vol.38(50), pp.16481-16490
- Publisher
- American Chemical Society
- DOI
- 10.1021/bi9919450
- PMID
- 10600109
- ISSN
- 0006-2960
- eISSN
- 1520-4995
- Language
- English
- Date published
- 12/14/1999
- Academic Unit
- Research Administration; Pharmaceutical Sciences and Experimental Therapeutics; Biochemistry and Molecular Biology; Chemistry
- Record Identifier
- 9984288719502771
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